a) Field of the Invention
The present invention relates to a sample vessel for X-ray microscopes, and more specifically to a vessel which is to be applied to microscopy using light sources emitting X-rays or soft X-rays and configured so as to be capable of containing samples to be observed together with an aqueous solution.
b) Description of the Related Art
In recent years remarkable progress has been made in research and development of light sources emitting X-rays and optical elements for X-rays. X-ray microscopes are offered as commercial products which have been obtained as one kind of systems developed by utilizing the progress. These X-ray microscopes are of various types which utilize imaging means such as a grazing incidence optical system of a Walter type (FIG. 1A), a Fresnel zone plate utilizing diffraction (FIG. 1B) and a direct incidence type Schwarzschild optical system comprising two spherical mirrors coated with multi-layer films (FIG. 1C).
Shown in FIG. 1A are Hyperboloids of revolution HR, reflecting surfaces RS, an object point OP, an Image point IP, and an ellipsoid of Revolution ER. Shown in FIG. 1B(2) are a Focal point of virtual Image VI, a Focal point of a real image RI, and a zone plate AP. Shown in FIG. 1C are an Image point Ip and an object point op.
It is known that these X-ray microscopes have spatial resolution which is ten times or more higher than that of the ordinary optical microscopes using the visible rays owing to a fact that the X-ray microscopes have limits of diffraction which are lowered in proportion to the wavelengths of X-rays. X-ray microscopes which use wavelengths of several hundred angstroms, for example, have merit in that these instruments permit us to expect to obtain resolution as high as 50 nanometers or so and, in addition, the development of compact plasma light sources which emit lasers having high luminance has accelerated the development of X-ray microscopes for laboratory use. Under these circumstances, X-ray microscopes are now regarded as excellent microscopes which are to be used in the future.
Further, attention is now being concentrated on X-ray microscopes using soft X-rays, i.e., soft X-ray microscopes, in addition to the X-ray microscopes which use the ordinary X-ray wavelength.
The soft X-ray microscopes feature merits in that they permit microscopy with resolution higher than that available with the optical microscopes, and in that they do not require, unlike electron microscopes, any pretreatment of samples to be observed. Moreover, since soft X-rays damage biological samples far less than electron beams, these rays are applicable to microscopes for observing biological samples with high resolution and in a non-colored state or in conditions where the samples are kept nearly in their living conditions as the occasion demands.
On the other hand, research is now being actively conducted for the utilization of soft X-rays which are within the so-called "window of water" region (soft X-rays having wavelengths within a range from 23 .ANG. to 44 .ANG.) since it has been clarified that biological samples can be observed in nearly living conditions thereof when soft X-rays within this region are used.
Now, description will be made of conventional examples of operating principles and configurations of soft X-ray microscopes with reference to FIG. 2, FIG. 3 and FIG. 4.
In the case of an imaging type soft X-ray microscope M which is illustrated in FIG. 2, a sample S to be observed contained in a sample vessel V is irradiated with soft X-rays which are emitted from a soft X-ray source LS and condensed by a condenser lens C, and a transmission image of the sample is imaged for detection by an objective lens O on a detector D. Since soft X-rays are absorbed remarkably by air, or have very low transmittance through air, a passage for the soft X-rays must be kept under vacuum and all members from the soft X-ray source LS to the detector D must be accommodated in a vacuum chamber VC. (A soft X-ray microscope of this type will hereinafter be referred to as a vacuum enclosed type.)
Further, a soft X-ray microscope of a type illustrated in FIG. 3 is used for microscopy of biological samples which cannot be kept under vacuum. In the case of this type of soft X-ray microscope, the vacuum chamber is divided into two vacuum chambers: a vacuum chamber VC.sub.1 which accommodates a section including the members from the soft X-ray source LS to the condenser lens C and has an exit window W.sub.1 disposed on an exit end face of this section; and another vacuum chamber VC.sub.2 which accommodates another section including members from the objective lens O to the detector D and has an entrance window disposed on an entrance end face of this section. A sample to be observed S contained in a sample vessel V is placed in a space d which is reserved so as to be as narrow as possible between the exit window emergence W.sub.1 and the entrance window W.sub.2 of the vacuum chambers VC.sub.1 and VC.sub.2 respectively that is, in a space AS which is opened to an atmosphere. (A soft X-ray microscope of this type will hereinafter be referred to as an atmosphere-open type.) In this type of soft X-ray microscope, the sample to be observed S is irradiated with soft X-rays which are emitted from the soft X-ray source LS and condensed by the condenser lens C, and emerge through the exit window W.sub.1. Soft X-rays which have transmitted through the sample to be observed S are received through the entrance window W.sub.2 and a transmission image of the sample S is imaged for detection by the objective lens O on the detector D.
When a sample, in particular a biological sample, is to be observed while it is kept in a wetted condition or while avoiding a drying of the sample under vacuum by using the soft X-rays, types of configurations of sample vessels applicable to the microscopy are different depending upon whether a soft X-ray microscope to be used is the vacuum enclosed type or the atmosphere-open type. When a soft X-ray microscope of the vacuum enclosed type is to be used for the microscopy, the sample to be observed must be kept in water under vacuum. In this case, the sample vessel V is configured so as to contain the sample to be observed S together with an aqueous solution AQ within an internal space IS which is reserved by forming two thin films F on a front surface and a rear surface of a relatively thin sample holding base plate B prepared so as to form the internal space. When a soft X-ray microscope of the atmosphere-open type which has a sample holding space open to atmosphere is to be used for the microscopy, it is unnecessary to form the thin films F on the sample base plate B, but it is sufficient to hold the sample to be observed S and the aqueous solution AQ in an internal space of the sample vessel V. As is judged from the fact described above that the soft X-rays are absorbed by an air layer formed in the sample holding space, however, it is necessary to configure the sample vessel so as to be capable of keeping the sample holding space under a pressure as low as possible.
Furthermore, FIG. 5 exemplifies a sample vessel which has been conventionally proposed. (See Japanese Patent Preliminary Publication No. Sho 63-298200.) This sample vessel V is configured as a pair of structures each of which consists of a silicon base plate SB and a thin film of silicon nitride F approximately 0.3 .mu.m thick formed on one surface of the silicon base plate SB, for example, by CVD method, and has an opening W which is to be used as an entrance window or an exit window and formed by anisotropically etching the other surface of the silicon base plate SB. A sample accommodating chamber CH is formed by cementing a spacer SP made of a material containing silicon and having a predetermined thickness to a surface of the thin film of silicon nitride F of one of the structures with a bonding agent containing silicon. Water containing biological samples is accommodated in the sample accommodating chamber CH and a surface of the thin film of silicon nitride F of the other structure is cemented to the spacer SP by using the same bonding agent so as to enclose the water containing the biological samples. The sample vessel V which has accommodated the water containing the biological samples and the sealed sample accommodating chamber as described above is set in a vacuum chamber and evacuated to a predetermined pressure when the vacuum enclosed type soft X-ray microscope is to be used for the microscopy. When the latter atmosphere-open type of soft X-ray microscopy is to be used, in contrast, the sample vessel is set in a space reserved between the exit window and the entrance window of the vacuum chambers preliminarily evacuated to predetermined pressure levels before effecting the microscopy using soft X-rays.
Each of the conventional sample vessels described above has a defect in that it allows the biological sample enclosed together with water to move freely during a period of time between when the sample is set in the microscope to when the microscopy starts, and during the observation of the sample. In other words, a slight movement of the biological sample hinders observation in case of X-ray microscopes and soft X-ray microscopes which have visual fields which are not as broad. The conventional sample vessels have a common defect in that they can hardly allow observation of a specific sample continuously for a long time since biological samples are moved due not only to convection of the water itself containing the biological samples, and activities of the cells of the biological samples, but also gravity and buoyancy applied to the biological samples in postures thereof.
On the other hand, even the soft X-rays which are within the "window of water" (a soft X-ray having a wavelength of 40 .ANG., for example) exhibits a transmittance of the order of 14% for a water layer 5 .mu.m thick, for example, which is not so high. It is therefore more advantageous to prepare a sample vessel so as to set a layer of an aqueous solution containing a biological sample thinner (in a direction in which soft X-rays transmit therethrough), but it is more difficult to prepare a sample vessel which sets a thinner layer of an aqueous solution. Overall transmittance is made very low when the sample vessel for the vacuum enclosed type soft X-ray microscope is applied with no modification to the atmosphere-open type soft X-ray microscope in which soft X-rays pass through at least one air layer formed between the exit window W.sub.1 and the entrance window W.sub.2 of the vacuum chambers VC.sub.1 and VC.sub.2 on respectively. Even in a case where the soft X-rays which are within the "window of water" region are to be used for microscopy, a problem lies in how the sample to be observed S and the aqueous solution AQ are held relative to the internal space IS when the thin films F sandwiching the sample holding base plate B are omitted in the sample vessel V.
Moreover, use of the sample vessel illustrated in FIG. 5 poses a problem which is described below. When a soft X-ray microscope adopts a white light source and a solid detector such as CCD's, the super-thin films which contain silicon and are used for composing the sample vessel allow transmission also of the visible rays and the detector detects the visible rays in addition to the desired soft X-rays, whereby the microscope cannot provide a microscopic image formed with the desired soft X-rays. In addition, when an X-ray source is a plasma light source emitting a laser, the sample vessel may be damaged by particles flying from the light source.